Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations.

BACKGROUND
Toxoplasma gondii, as a eukaryotic parasite of the phylum Apicomplexa, can infect almost all the warm-blooded animals and humans, causing toxoplasmosis. Rhoptry neck proteins (RONs) play a key role in the invasion process of T. gondii and are potential vaccine candidate molecules against toxoplasmosis.


METHODS
The present study examined sequence variation in the rhoptry neck protein 10 (TgRON10) gene among 10 T. gondii isolates from different hosts and geographical locations from Lanzhou province during 2014, and compared with the corresponding sequences of strains ME49 and VEG obtained from the ToxoDB database, using polymerase chain reaction (PCR) amplification, sequence analysis, and phylogenetic reconstruction by Bayesian inference (BI) and maximum parsimony (MP).


RESULTS
Analysis of all the 12 TgRON10 genomic and cDNA sequences revealed 7 exons and 6 introns in the TgRON10 gDNA. The complete genomic sequence of the TgRON10 gene ranged from 4759 bp to 4763 bp, and sequence variation was 0-0.6% among the 12 T. gondii isolates, indicating a low sequence variation in TgRON10 gene. Phylogenetic analysis of TgRON10 sequences showed that the cluster of the 12 T. gondii isolates was not completely consistent with their respective genotypes.


CONCLUSION
TgRON10 gene is not a suitable genetic marker for the differentiation of T. gondii isolates from different hosts and geographical locations, but may represent a potential vaccine candidate against toxoplasmosis, worth further studies.


Introduction
oxoplasmosis caused by Toxoplasma gondii is one of the most common parasitic zoonoses worldwide, with a wide range of hosts including almost all warmblooded animals (1)(2)(3)(4). Global epidemiologic studies of toxoplasmosis indicate that overall 33% people infected the T. gondii (1,5).
The rhoptry is a subcellular organelle of apicomplexan parasites. Rhoptry neck proteins (RONs) are secreted by rhoptry for the formation of moving junction (MJ), which plays an important role in the invasion of T. gondii (6). Therefore, the research on RONs can help us to better understand the pathogenic mechanism of T. gondii and explore the effective approaches for prevention and treatment of toxoplasmosis. Some studies indicate that RONs are concerned with T. gondii invasion, so they are underlying candidate antigens of DNA vaccine to against T. gondii (7). Rhoptry neck protein 10 (TgRON10) is a component of the newly identified RON9/RON10 complex in T. gondii, related with development of T. gondii in intestinal epithelial cells (8).
However, little is known about sequence variation in TgRON10 gene among T. gondii isolates of different genotypes. The aim of this study was to examine the sequence variation in TgRON10 genes among T. gondii isolates from different hosts and geographical locations, and to assess whether the TgRON10 gene sequence may represent a new marker for studying T. gondii population genetic structures.

T. gondii isolates
Ten T. gondii isolates originating from different hosts and geographical locations were used in this study from Lanzhou Province during 2014 (Table 1), and genomic DNA (gDNA) of these T. gondii isolates was prepared and genotyped in our previous studies (9)(10)(11)(12). Two corresponding sequences of strains ME49 (ToxoDB: TGME49_261750) and VEG (ToxoDB: TGVEG_261750) were obtained from the ToxoDB database.

Sequencing of the TgRON10 amplicons
Positive TgRON10 amplicons were purified using the spin columns according to the manufacturer's recommendations (Wizard™ PCR-Preps DNA Purification System, Promega, USA), ligated into pGEM-T-Easy vector (Promega), and then transformed into the JM109 competent cells (Promega, USA). Following the screening by PCR amplification, the positive colonies were sequenced by Shanghai Songon Biological Engineering Biotechnology Company.

Sequence analysis and phylogenetic reconstruction
The obtained TgRON10 gene sequences from different T. gondii strains were aligned using the computer program ClustalX 1.83 (13), and sequence variation was determined among the examined T. gondii strains. Phylogenetic reconstructions based on the complete sequences of TgRON10 gene among different T. gondii strains was performed by Bayesian inference (BI) and maximum parsimony (MP) using Neospora caninum (GenBank accession No. FR823389.1) as the out-group. BI analyses were conducted with four independent Markov chains run for 10000000 metropoliscoupled MCMC generations, sampling a tree every 10000 generations in MrBayes 3.1.1 (14). The first 250 trees were omitted as burn-ins and the remaining trees were used to calculate Bayesian posterior probabilities (PP). MP analysis was performed using PAUP* 4.0b4a (15), with indels treated as missing character states. Overall, 1000 random addition searches using TBR were performed for each MP analysis. Bootstrap probability (BP) was calculated from 1000 bootstrap replicates with 10 random additions per replicate in PAUP. Phylograms were drawn using the Tree View program ver. 1.66 (16).

Results
PCR amplification of TgRON10 gene from different T. gondii isolates produced a single band of approximately 4600 bp in length on agarose gel (Fig. 1). Positive TgRON10 amplicons were purified and ligated with clone vector, and then transformed into the competent cells. Following the screening by PCR amplification, the positive colonies were sequenced from both directions. The obtained entire genomic sequences of TgRON10 gene was 4759 bp in length for the strains CTG and VEG, 4762 bp for the strains GT1, MAS, RH, SH and PYS, 4763 bp for the strain TgCatBr5, and 4760 bp for the other four strains. Analysis of all the 12 TgRON10 complete genomic sequences revealed 7 exons and 6 introns in the TgRON10 gene, the A+T contents varied from 48.43% to 48.61% in the entire sequence. There were 124 nucleotide position variations in the entire genomic sequences (Fig. 2). A total of 55 nucleotide position variations in exons with a distribution of two deletions of 3 bp in the sequence of strains CTG and VEG, 40 transitions (C<->T, A<->C, and A<->G) and 9 transversions (A<->T and C<->G) (R=transition/transversion=4.4) ( Table 2). In addition, there were 124 nucleotide position variations in the intron among the 12 examined T. gondii isolates, including 42 deletions, 75 transitions (C<->T, T<->G, A<->C, and A<->G) and 7 transversions (A<->T and C<->G) ( Table 3).

Discussion
In the present study, the alignment of TgRON10 entire genomic sequences showed that sequence variation were 0-0.6% in all examined strains. The deduced amino acid sequence analysis showed the presence of 30 substitutions and two deletions among the 12 examined T. gondii isolates, which is lower than that in ROP7 and ROP13 genes (17,18). Variation in TgRON10 sequences among the examined T. gondii isolates was slightly low, and similar results were found in previous studies, such as PLP1 (19), MIC13 (20) and other genes among the clonal lineages of T. gondii (21). In summary, our data indicated the existence of low sequence variation in TgRON10 gene among different T. gondii isolates, thus it is not a suitable genetic marker for genotyping studies in T. gondii. However, due to the high identity in different T. gondii isolates, RON10 gene may be an ideal immune effector molecule against different T. gondii isolates infection, worth further study. Phylogenetic analysis using BI and MP based on TgRON10 sequence of all 12 T. gondii strains has shown that the two major clonal lineages (Type I and III) can be differentiated (Fig. 3). All the Type I strains SH, GT1, and RH clustered together. Two Type III strains CTG and VEG grouped together. However, the two Type II strains PRU and ME49 were separated strains representing other genotypes (Fig. 3).

Conclusion
This study revealed the existence of low sequence variability in TgRON10 gene among the examined T. gondii isolates from different hosts and geographical locations. TgRON10 gene may not be a suitable marker for population genetic studies of T. gondii isolates but may represent a potential vaccine candidate against T. gondii infection.